1. Field of the Invention
The present invention relates to the fields of cardiology, ischemic heart disease and cardiovascular pharmacology as well as diabetes and cancer. More specifically, the present invention relates to, inter alia, peptide modulators of the “d” subunit of F1Fo ATP synthase/ATPase (d F1Fo and uses thereof.
2. Description of the Related Art
In the United States, someone dies every minute as a result of coronary artery blockage resulting in a heart attack (1). In fact, heart attacks are responsible for more annual deaths than any other single medical condition and half of those suffering a myocardial infarction (MI) will not survive the attack (1). Of those arriving at the hospital alive, 40% will die within the first year, and for those surviving past 1 year, many will develop co-morbidities such as congestive heart failure, which itself carries a 50% mortality rate at 5 years. The most common underlying cause of heart attacks involves occlusion of one or more coronary vessels by atherosclerosis or thrombi formation. This prevents the flow of blood, and consequently the supply of oxygen, nutrients, and other factors to the myocardium. If blood flow is not restored promptly, massive cell death occurs resulting in permanent cardiac injury. In diabetics, the progression of coronary artery disease is greatly accelerated and the severity of cardiac injury suffered following a heart attack is generally 3-5-times as severe as in non-diabetics of comparable age (2).
Clinical therapy for heart attack victims focuses on the rapid restoration of blood flow by thromobolysis, angioplasty, stenting and when appropriate surgical coronary artery bypass grafts (3). A majority of cardiac cell death associated with a heart attack actually occurs during the early phases of reperfusion when blood flow is restored (4). Therefore, cardioprotective agents that minimize cell death during reperfusion therapy are desperately needed and would greatly improve the outcomes of these patients.
Mammalian protein kinase C (PKC) exists as a 10 member family of closely related serine/threonine kinases, with each individual kinase being identified by a unique Greek letter designation. δPKC is a member of this large family of enzymes. PKC isozymes have been categorized into 3 subfamilies: classical (α, βI, βII, γ), novel (δ, ε, η, θ) and the atypical (ζ, λ/t) PKC based on amino acid homologies and responses to various PKC activators (5). In terms of in vitro phosphotransferase activities, the classical PKCs are activated by Ca2+, phosphatidylserine (PS), and 1,2, sn-diacylglycerol (DG). The novel PKCs are Ca2+-independent and DG/PS-sensitive, while the atypical PKCs are insensitive to both Ca2+ and DG. In many cases the mitochondrial lipid cardiolipin can also activate PKC isozymes (6). In general, each PKC isozyme has a regulatory domain (which contains the PKC activator binding sites and many subcellular localization domains), and a catalytic domain (which contains the ATP-binding and phosphotransferase sites).
PKC structure can be divided into 5 variable amino acid sequence regions (which differ between isozymes) and 5 conserved amino acid regions. The variable amino acid regions may play important roles in PKC isozyme-unique subcellular targeting and functions in vivo (7,8). Studies in cardiac myocytes, and many other cell types, have demonstrated that upon simultaneous activation of PKC isozymes, each enzyme can translocate to different subcellular sites (9,10). This differential targeting is thought to be mediated by PKC anchoring proteins known as Receptors for Activated C-Kinase (RACKS) (7,8). In the simplest form of this model each PKC isozyme has its own RACK and only that isozyme can bind to its' own RACK because the other PKC isozymes lack the crucial structural determinants (e.g. amino acid sequence) for binding. This provides a mechanism whereby different PKC isozymes can have isozyme-selective regulatory functions within the same cell. For example, a PKC isozyme translocating to the cell nucleus might regulate gene expression, whereas a different PKC isozyme may translocate to mitochondria to regulate energy production.
Cardiac IR injury occurs when blood flow to the heart is impaired (ischemia) and when normal blood flow is restored immediately after a heart attack (reperfusion), εPKC has cardioprotective actions against IR injury through a process known as cardiac ischemic preconditioning (PC) (16). PC is a paradoxical response whereby brief bouts of ischemia and reperfusion produce protection against a subsequent more sustained IR insult (17). The role of the PKC isozyme δPKC in cardioprotection and damage is more controversial with reports indicating it plays significant roles in both PC and IR injury. For example, Mayr et al., reported that δPKC knockout mice demonstrated decreased glycolysis and an increased lipid metabolism, which uses more oxygen to make energy, under baseline conditions, and were unable to induce a cardiac PC response (24,25). In contrast, Mochly-Rosen and colleagues demonstrated that the activation of δPKC induces apoptosis and delays the reactivation of pyruvate dehydrogenase during IR injury which slows the re-supply of acetyl CoA to the Kreb's cycle (12,14). δPKC has also been reported to translocate to the mitochondria and interacts with the proapoptotic protein Bad to induce pathological hypertrophy and cardiac apoptosis (12).
There have been studies implicating PKC isozymes in the pathology of diabetes in heart and other tissues. The PKC activating lipid DG is elevated in diabetic myocardium (27-29) and reduction of DG levels appears to attenuate diabetic effects on the heart (29). Similarly, there have been reports of elevated cardiac PKC isozyme expression (30-32), translocation (33) and activity (34) under hyperglycemic conditions. Studies suggest that hyperglycemia-induced translocation of the α, β, ε, or δPKC isozymes correlates with phosphorylation of cardiac troponin I (cTnI) which may contribute to impaired diastolic relaxation and loss of myofibrillar Ca2+ sensitivity (33,35). In addition, excessive PKC isozyme-modulation of ryanodine receptors (36-38), the Na/Ca2+ exchanger (39), and other Ca2+ handling proteins has been reported in diabetic myocardium. εPKC-mediated hyperphosphorylation of connexin 43 (Cx43) may contribute to Cx43 proteosomal degradation and cardiac arrhythmias in diabetic hearts (40,41). Farese et al. demonstrated that muscle-specific knock-out of ζPKC correlated with defective translocation of the GLUT4 glucose transporter to the plasma membrane and the development of insulin resistance in adipocytes (42). Finally, the βPKC isozymes have been implicated in hyperglycemia-induced hypertrophy (43), elevation of ROS (43), and diabetic cardiomyopathy (44). Therefore, an extensive literature supports a role for PKC isozymes in the cardiac pathology of diabetes, but few studies have examined mitochondrial PKC isozyme targets in diabetes. Malhotra et al. reported that transgenic over-expression of an εPKC-selective activating peptide reduced streptozotocin (STZ)-induced εPKC translocation to the plasma membrane and mitochondria, which was associated with diminished oxidative stress, ventricular dysfunction, and apoptosis (45). Arikawa et al. used oligo-nucleotide arrays to correlate up-regulation of cardiac PKC isozyme gene expression with diminished levels of pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) and the mitochondrial uncoupling protein 3 (UCP3) (46). ATP levels and OXPHOS enzyme activities are reduced in diabetes (47-58).
The mammalian F1Fo ATP synthase is a 16 subunit enzyme complex. It contains an F1 domain (3α, 3β, γ, δ, and ε subunits), which protrudes into the mitochondrial matrix (59-61). The interlaces between α and β subunits are the site of nucleotide binding and ATP synthesis. It also has an Fo domain, which is a proton channel that traverses the IM and allows proton re-entry into the mitochondrial matrix down a concentration gradient. This proton movement provides the energy for ATP synthesis (60,62,63,65). The F1 and Fo domains are connected by a central stalk consisting of the γ, δ, and ε subunits and by a peripheral stalk, which is made up of the OSCP, F6, b, and d subunits (59-61). The central stalk is thought to rotate along with the c subunits during ATP synthesis. This rotation is crucial for proton movement through the Fo domain. The peripheral stalk acts as a stator to prevent the α and β subunits from rotating with the central stalk and c subunits. This appears to be crucial for the phosphorylation of ADP to ATP on the α and β subunits.
Following severe cardiac IR injury ATP levels decline substantially (13,64). A major component of this drop involves the loss of the electrochemical/proton gradient across the IM, which supplies the energy for ATP production by F1Fo ATP synthase. Therefore, shortly after the induction of ischemia the enzyme becomes inhibited. It then makes a futile attempt to re-establish the mitochondrial IM potential by operating in reverse to pump protons out of the mitochondrial matrix. This process is very inefficient and requires energy which is supplied by the F1Fo complex then operating in reverse-mode as an ATPase (65). If ischemia is not interrupted, F1Fo ATPase activity will contribute heavily to the loss of cardiac ATP (65). The activity of the F1Fo complex is also regulated by two endogenous inhibitors: inhibitor of F1 (IF1) and Ca2+-sensitive binding-inhibitor protein (CaBl) (62, 65-69). When the enzyme is in ATPase-mode its activity is thought to be partially limited by the IF1 protein.
In support of this, IF1 binds F1Fo ATPase at the α and β subunit interface (65), under conditions of decreased pH and mitochondrial membrane potential (65), such as would occur in ischemia. The role of CaBl is less clear. It binds to the enzyme under low mitochondrial intracellular Ca2+ concentration and is released from the enzyme following an increase in mitochondrial Ca2+ concentration (68). Therefore, as Ca++ increases in the cell to facilitate increased contractility, mitochondrial Ca++ also increases. This relieves the inhibition of F1Fo ATP synthase by CaBl to allow more ATP synthesis as necessary for the increased cardiac contractility. Presumably its inhibition would be relieved during IR injury also, since calcium overload of cardiac myocyte mitochondria occurs in IR injury. It is generally agreed however, that changes in mitochondrial inner membrane potential and IF1- and CaBl-mediated inhibition of F1Fo activities cannot completely account for the regulation of the F1Fo enzyme complex. In addition to the F1Fo ATPase-mediated ATP hydrolysis in cardiac IR injury, the return of aerobic ATP synthesis is also impaired (13,65) and the heart attempts to compensate by utilization of glucose as a preferred substrate (instead of predominately fatty acids) in anaerobic glycolysis (65). Anaerobic ATP production is not sufficient to satisfy the intense cardiac energy demands required to support contractility indefinitely and other functions. It also generates lactic acid with consequent lactic acidosis, which further damages the heart and inhibits/impairs glycolytic enzymes themselves (65). Therefore, enhancing the return of aerobic ATP production following cardiac IR would improve the survival and functionality of the heart.
Diabetes induces both structural and functional changes in cardiac mitochondria including significant loss of proteins involved in OXPHOS (47-55). There are also losses in mitochondrial DNA, Ca2+ uptake, creatinine phospho-kinase (CPK), and ATP synthase activities (37,50,54-57) which translate into lower myocardial ATP levels. The healthy, non-diabetic heart generates ATP mostly from oxidation of fatty acids (FA) (˜70%) and to a lesser extent from glucose (25%), lactate and other sources (5%) (58). In hyperglycemic states such as diabetes, excessive amounts of free FA are liberated and there is an even greater reliance on FA and a reduced utilization of glucose for cardiac energy (47,50). This increase in FA levels induces peroxisome proliferator-activated receptors (PPARs) and their cofactor peroxisome proliferator-activated receptor cofactor 1-α(PGC1-α) to enhance the transcription of genes coding for proteins involved in virtually all aspects of FA utilization (50). This leads to greater β-oxidation of long chain FA and an increase in electrons (NADH and FADH2) entering the electron transport chain (ETC). However, decreased levels of OXPHOS proteins could contribute to a greater frequency of electron leak from ETC complexes and contribute to a chronic increase in ROS production, which can cause oxidative damage to proteins, lipids, and nucleic acids producing further damage in diabetes.
The yield of ATP per oxygen atom consumed indicates that oxidation of FA requires more oxygen than glucose oxidation, which may contribute to decreased cardiac efficiency in diabetic hearts. One mechanism promoting this inefficiency is the progressive FA-induced uncoupling of respiration by a family of proton translocases in the IM known as mitochondrial uncoupling proteins (UCPs). Cardiac expression of UCP2 and UCP3 is thought to be up-regulated by elevated levels of FA (49,52,58,70) and UPC expression appears to be induced by elevation of superoxide (71). Enhanced UCP expression is thought to uncouple respiration by disrupting membrane potential through proton leakage across the IM. In addition, there may be other proteins which uncouple respiration under diabetic states such as the adenine nucleotide transporter in the IM (72). This indicates that there is a lower ATP/oxygen ratio in diabetic hearts. Interestingly, studies by Boudina et al. demonstrated that increased UCP activity resulted in mitochondrial uncoupling in db/db diabetic mice (73). When compared to wild type mice, db/db mice showed increased respiration in the presence of oligomycin, decreased ATP production, and decreased ATP/oxygen ratios. An increased respiration in the presence of oligomycin would favor superoxide generation from the ETC. This is interesting in the context of these results because inhibition of F1Fo ATP synthase via the δPKC-dF1Fo interaction may also increase mitochondrial ROS production. In addition, δPKC has been reported to be a major player in cardiac IR injury and has been shown to elevate mitochondrial ROS production and induce apoptosis (15, 74-76). δPKC may therefore contribute to the exacerbation of cardiac injury in diabetes by chronically reducing ATP levels via a previously uncharacterized inhibition of the F1Fo ATP synthase complex.
Thus, there is a continued need in the art for identification of compositions and methods for treating, among other things, ischemia/reperfusion disorders. The present invention fulfills this long-standing need and desire in the art.